1. Field of the Invention
The present invention relates to a novel member of the vanilloid receptor family. More specifically, the present invention relates to the discovery, identification and characterization of nucleotides that encode vanilloid receptor-2 (VR2), a receptor having homology with the rat vanilloid receptor-1 protein. The invention encompasses VR2 polynucleotides, host cell expression systems, VR2 polypeptides (including fragments, variants, derivatives and analogs thereof), VR2 fusion proteins, antibodies to VR2, agonists and antagonists of VR2, and other compounds that modulate VR2 gene expression or VR2 activity, that can be used for diagnosis, drug screening, and treatment or prevention of disorders which include, but are not limited to, chronic pain syndromes, congenital pain insensitivity, inflammation, ischemia, host defense dysfunction, immune surveillance dysfunction, arthritis, multiple sclerosis, autoimmunity, immune dysfunction, and allergy.
2. Related Art
The concentration of free Ca+2 in the cytosol of any cell is extremely low (≈10xe2x88x927 M), whereas the concentration of free Ca+2 in the extracellular fluid (≈10xe2x88x923 M) and in the endoplasmic reticulum is quite high. Thus, there is a large gradient tending to drive Ca+2 into the cytosol across both the plasma membrane and the endoplasmic reticulum membrane. When a signal transiently opens Ca+2 channels in either of these membranes, Ca+2 rushes into the cytosol, dramatically increasing the local Ca+2 concentration and triggering Ca+2-responsive proteins in the cell. Ca+2 has been demonstrated to act as an intracellular messenger in a wide variety of cellular responses, such as, for example, transmission of an action potential in nerve cells, muscular contraction, and cell secretion, activation, survival, proliferation, migration, and differentiation.
Pain is initiated when a subgroup of sensory neurons, called nociceptors, are activated by noxious chemical, thermal or mechanical stimuli. The activated nociceptors convey information regarding the noxious stimuli to pain processing centers in the spinal cord and brain (Fields, H., Pain (McGraw-Hill, New York, 1987). Nociceptors are characterized in part, by their sensitivity to vanilloids (i.e., chemicals containing vanillyl groups), such as, for example, capsaicin, the main pungent ingredient in capsaicin peppers. In mammals, exposure of nociceptor terminals to capsaicin leads to excitation of the neuron and the consequent perception of pain and local release of inflammatory mediators. Prolonged exposure of nociceptor terminals to capsaicin leads to the desensitization of these neurons to capsaicin and other noxious stimuli (Szolcsanyi, Y., in Capsaicin and the Study of Pain (ed. Wood J.) 255-272 (Academic, London, 1993). This phenomenon of desensitization has led to the use of capsaicin as an analgesic agent in the treatment of painful disorders ranging from viral and diabetic neuropathies to rheumatoid arthritis (Campbell, E., in Capsaicin and the Study of Pain (ed. Wood J.) 255-272 (Academic, London, 1993; Szallasi et al., Pain 68:195-208 (1996)).
Recently, a cDNA encoding vanilloid receptor subtype-1 (VR1), has been isolated from a rodent dorsal root ganglion plasmid cDNA library (Caterina et al., Nature 389:816-824 (1997). This clone encodes a polytopic integral membrane protein containing six transmembrane domains, four extracellular domains, four intracellular domains, and an additional short hydrophobic region between transmembrane domains five and six that may contribute to an ion permeation path (Hardie et al., Trends Neurosci. 16:371-376 (1993)). The product of this clone is a calcium permeable, non-selective cation channel that is structurally related to members of the TRP family of ion channels (see, e.g., Montell et al., Neuron 2:1313-1333 (1989); and Hardie et al., Trends Neurosci. 16:371-376 (1993)).
Capsaicin binding to VR1 has been demonstrated to trigger an increase in intracellular free calcium. Additionally, transfection of VR1 into non-neuronal cells has been shown to induce cytotoxicity upon continuous exposure to capsaicin. These observations are consistent with necrotic cell death resulting from excessive ion influx.
VR1 is activated (i.e., the VR1 cation-selective channel is opened), by capsaicin, capsaicin agonists, and other vanilloid compounds (e.g., resiniferatoxin), and antagonized by capsaicin antagonists (e.g., capsazepine and ruthenium red). Further, hydrogen ions potentiate the response of VR1 to low concentrations of capsaicin: thus, VR1 may be involved in the detection of noxious stimuli that accompany such conditions as inflammation and ischemia (Caterina et al, Nature 389:819-824 (1997).
Additionally, VR1 is activated when ambient temperatures are elevated to elicit pain in humans or pain associated behaviors in animals, indicating that, in addition to its role in transducing noxious chemical stimuli, VR1 functions as a transducer of painful thermal stimuli in vivo (Caterina et al., Nature 389:816-824 (1997).
The involvement of a vanilloid receptor family member in transducing thermal and chemical stimuli suggests that members of this family of cation channels are involved in diverse human disease states ranging from congenital pain insensitivity, to chronic pain syndromes and more generally that members of this family mediate cellular responses such as cell secretion, activation, survival, proliferation, migration and differentiation; that vanilloid receptor family members provide an important model system for the in vitro study of hyperalgesia; and that vanilloid receptors provide defined targets for the development of new analgesic agents.
The present invention provides isolated nucleic acid molecules comprising polynucleotides encoding a VR2 receptor having the amino acid sequence shown in FIGS. 1A-1D (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone encoding the VR2 receptor deposited in a vector as ATCC Deposit Number 263082 on Jul. 30, 1998. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them or other genetically modified host cells to produce VR2 polypeptides (including fragments, variants, derivatives, and analogs thereof) by recombinant techniques.
The invention further provides isolated VR2 polypeptides having amino acid sequences encoded by the polynucleotides described herein.
The present invention also provides a screening method for identifying compounds capable of eliciting a cellular response induced by VR2, which involves contacting cells which express VR2 with the candidate compound, assaying a cellular response (e.g., ion flux, such as, Ca+2 flux), and comparing the cellular response to a standard cellular response, the standard being assayed in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by VR2 receptors, which involves contacting cells which express VR2 receptors with the candidate compound in the presence of a VR2 agonist (e.g., a vanilloid compound, such as capsaicin) or other stimulus (e.g., thermal stimuli), assaying a cellular response (e.g., ion flux, such as, Ca+2 flux), and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made between the agonist and VR2 or when VR2 is exposed to the stimulus, in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an agonist
In another embodiment, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on the binding of cellular ligands (e.g., vanilloid compounds, such as, capsaicin) to VR2. In particular, the method involves contacting VR2 with a ligand or other stimulus (e.g., thermal stimuli) and a candidate compound and determining whether ligand binding to the VR2 receptors is increased or decreased due to the presence of the candidate compound.
The invention further provides a diagnostic method useful during diagnosis or prognosis of disease states resulting from aberrant pain sensitivity, or aberrant cell secretion, activation, survival, migration, differentiation and/or proliferation, due to alterations in VR2 coding sequences and/or receptor expression.
An additional embodiment of the invention is related to a method for treating an individual in need of an increased level of VR2 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of VR2 polypeptides or polynucleotides of the invention or a VR2 agonist.
A still further embodiment of the invention is related to a method for treating an individual in need of a decreased level of a VR2 receptor activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of VR2 polypeptides or polynucleotides of the invention a VR2 antagonist.
The invention additionally provides soluble forms of the polypeptides of the present invention. Soluble peptides are defined by amino acid sequences wherein the sequence comprises the polypeptide sequence lacking a transmembrane domain (e.g., VR2 polypeptide fragments corresponding to intracellular and/or extracellular domains). Such soluble forms of the VR2 receptor are useful as antagonists of the membrane bound forms of the receptor.